Éverton Fagonde da Silva

Characterization of virulence of Leptospira isolates in a hamster model

Effort has been made to identify protective antigens in order to develop a recombinant vaccine against leptospirosis. Several attempts failed to conclusively demonstrate efficacy of vaccine candidates due to the lack of an appropriate model of lethal leptospirosis. The purposes of our study were: (i) to test the virulence of leptospiral isolates from Brazil, which are representative of important serogroups that cause disease in humans and animals; and (ii) to standardize the lethal dose 50% (LD(50)) for each of the virulent strains using a hamster (Mesocricetus auratus) model. Five of seven Brazilian isolates induced lethality in a hamster model, with inocula lower than 200 leptospires. Histopathological examination of infected animals showed typical lesions found in both natural and experimental leptospirosis. Results described here demonstrated the potential use of Brazilian isolates as highly virulent strains in challenge experiments using hamster as an appropriate animal model for leptospirosis. Furthermore these strains may be useful in heterologous challenge studies which aim to evaluate cross-protective responses induced by sub-unit vaccine candidates.

Recombinant vaccines against Leptospirosis

Leptospirosis is an important neglected infectious disease that occurs in urban environments, as well as in rural regions worldwide. Rodents, the principal reservoir hosts of pathogenic Leptospira spp., and other infected animals shed the bacteria in their urine. During occupational or even recreational activities, humans that come into direct contact with infected animals or with a contaminated environment, particularly water, are at risk of infection. Prevention of urban leptospirosis is largely dependent on sanitation measures that are often difficult to implement, especially in developing countries. Vaccination with inactivated whole-cell preparations (bacterins) has limited efficacy due to the wide antigenic variation of the pathogen. Intensive efforts towards developing improved recombinant vaccines are ongoing. During the last decade, many reports on the evaluation of recombinant vaccines have been published. Partial success has been obtained with some surface-exposed protein antigens. The combination of protective antigens and new adjuvants or delivery systems may result in the much-needed effective vaccine.

Protection against lethal leptospirosis after vaccination with LipL32 coupled or coadministered with the B subunit of Escherichia coli heat-labile enterotoxin.

ABSTRACT Leptospirosis, a worldwide zoonosis, lacks an effective, safe, and cross-protective vaccine. LipL32, the most abundant, immunogenic, and conserved surface lipoprotein present in all pathogenic species of Leptospira, is a promising antigen candidate for a recombinant vaccine. However, several studies have reported a lack of protection when this protein is used as a subunit vaccine. In an attempt to enhance the immune response, we used LipL32 coupled to or coadministered with the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) in a hamster model of leptospirosis. After homologous challenge with 5× the 50% lethal dose (LD50) of Leptospira interrogans, animals vaccinated with LipL32 coadministered with LTB and LTB::LipL32 had significantly higher survival rates (P < 0.05) than animals from the control group. This is the first report of a protective immune response afforded by a subunit vaccine using LipL32 and represents an important contribution toward the development of improved leptospirosis vaccines.

High rates of serological response to a modified hepatitis B vaccination schedule in HIV-infected adults subjects

We evaluated a modified HBV regimen in a cohort of HIV-infected subjects in Rio de Janeiro, Brazil. HIV-infected subjects with no serologic evidences of previous hepatitis B infection were immunized with 4 doses (40 microg each) of recombinant hepatitis B vaccine given at 0, 1, 2 and 6 months. Blood samples were collected 1 month after the last dose and anti-HBs titers were measured. A protective antibody response was defined as an anti-HBs titer >or=10 mIU/mL. Forty-seven subjects (30 women, 17 men; mean age was 36 years, ranging from 21 to 58 years) were included in the final analysis. Median baseline CD4+ lymphocyte count was 402 cells/mm(3) and 33 subjects (70%) had an HIV viral load below 80 copies/mL. A protective antibody response was observed in 42 (89%) subjects. Thirty-seven (78%) and 28 (60%) patients developed anti-HBs titers higher than 100 mIU/mL and 1000 mIU/mL, respectively. 1 out of 5 non-responders (20%) had an HIV viral load below the detection limit, in contrast with 32 (76%) of those with an adequate serologic response (p=0.02). These findings suggest that 4-double dose alternative schedule may be considered to overcome the lower seroconversion rates observed with the standard regimens in HIV-infected subjects.

Vaccination against hepatitis B with 4-double doses increases response rates and antibodies titers in HIV-infected adults

BACKGROUND Antibody responses to standard regimens of hepatitis B (HBV) vaccination are lower in HIV-infected subjects and the best hepatitis B vaccine schedule in this population is not known. OBJECTIVE To assess the immunogenicity and to evaluate predictors of serologic response of a modified regimen of a HBV recombinant vaccine in a cohort of HIV-infected subjects. METHODS HIV-infected subjects received 4 doses (40 μg) of a recombinant HBV vaccine at 0, 1, 2 and 6 months. Demographic information as well as CD4 cell count and plasma viral load were assessed at baseline. Protective and strong responses were defined as an anti-HBs titer ≥10 mIU/mL and ≥100 mIU/mL, respectively and were evaluated one month after the third and the fourth doses. RESULTS 163 HIV-infected individuals were evaluated 67 (40%) were male and median age was 37 years. Median CD4 cell count was 385 cells/mm(3) and 113 (70%) had undetectable HIV-1 viral load. Protective antibody response was observed in 83 and 91% and a strong antibody response was observed in 62 and 80% of the subjects after 3 and 4 doses, respectively. In a multivariate logistic model undetectable HIV-1 viral load and higher CD4 cell counts were independent predictors of a strong antibody response after 4 doses. Patients with undetectable HIV viral load were almost 3 times more likely to have anti-HBs titers above 100 mIU/mL than those with detectable viral load. CONCLUSIONS A 4-double-dose regimen of a recombinant HBV vaccine increased response rates and determined higher antibody titers which may translate in prolonged protection against HBV. Inclusion of a fourth dose of HBV vaccine for HIV-infected subjects should be considered in the public health setting.

Leptospira noguchii and human and animal leptospirosis, Southern Brazil.

To the Editor: Pathogenic leptospires, the causative agents of leptospirosis, exhibit wide phenotypic and genotypic variations. They are currently classified into 17 species and >200 serovars (1,2). Most reported cases of leptospirosis in Brazil are of urban origin and caused by Leptospira interrogans (3). Brazil underwent a dramatic demographic transformation due to uncontrolled growth of urban centers during the last 60 years. Urban slums are sites of poor sanitation that favors rat-borne transmission of leptospirosis among humans. Thus, this may explain the major involvement of serovar Copenhageni (L. interrogans). The predominance of L. interrogans is likely due to the underestimation of rural cases of leptospirosis.

Subunit Approach to Evaluation of the Immune Protective Potential of Leptospiral Antigens

ABSTRACT Leptospirosis is the most widespread zoonosis in the world. Current vaccines are based on whole-cell preparations that cause severe side effects and do not induce satisfactory immunity. In light of the leptospiral genome sequences recently made available, several studies aimed at identification of protective recombinant immunogens have been performed; however, few such immunogens have been identified. The aim of this study was to evaluate 27 recombinant antigens to determine their potential to induce an immune response protective against leptospirosis in the hamster model. Experiments were conducted with groups of female hamsters immunized with individual antigen preparations. Hamsters were then challenged with a lethal dose of Leptospira interrogans. Thirteen antigens induced protective immune responses; however, only recombinant proteins LIC10325 and LIC13059 induced significant protection against mortality. These results have important implications for the development of an efficacious recombinant subunit vaccine against leptospirosis.

Monitoring Leptospira strain collections: the need for quality control

The purpose of this study was to perform a 16S sequence-based quality control of two Leptospira strain collections. 16S rRNA gene sequencing was used to verify two Leptospira reference collections provided by the World Health Organization and maintained at a reference laboratory for leptospirosis in Brazil. Among the 89 serovars evaluated, four conflicting strains were identified in one of the collections. Although 16S rRNA gene sequencing cannot identify Leptospira beyond the species level, it is suitable for the identification of contamination and quality control of leptospiral reference collections. This study highlights the importance of the availability of high-quality 16S rRNA sequences in public databases. In addition, it emphasizes the need for periodical verifications and quality control of Leptospira reference collections.

Avaliação da modulação autonômica da freqüência cardíaca nas posturas supina e sentada de homens jovens sedentários

OBJECTIVE: To evaluate and compare the autonomic heart rate (HR) modulation, under resting conditions in relation to body posture, in sedentary young adults. METHODS: Twenty young healthy and sedentary men aged 22.6 ± 2.5 years participated in the study. The HR and R-R intervals (in ms) of the electrocardiogram (EKG) were obtained in real time using the modified DII derivation, with the volunteers at rest in the supine and seated positions, for 15 minutes. The R-R data were analyzed in the time domain, by means of the RMSSD, RMSM and pNN50 (%) indices; and in the frequency domain, by means of spectral analysis and fast Fourier transforms (FFT), using low frequency (LF) and high frequency (HF) bands expressed as normalized units and as the LF/HF ratio. The statistical analysis consisted of the Spearman test for correlation analyses and the Wilcoxon test for paired samples, with significance of a= 5%. RESULTS: In the time domain, the RMSSD and pNN50 indices demonstrated statistically significant differences between the supine and seated positions (p 0.05). In the frequency domain, the LF and HF bands and the LF/HF ratio demonstrated statistically significant differences between the supine and seated positions (p< 0.05). CONCLUSIONS: The results demonstrated that, by changing the posture, autonomic adjustments were produced to the parasympathetic and sympathetic nervous systems with regard to HR control. This can be attributed to the integrity of the neurocardiac system.

Dried blood spot samples: optimization of commercial EIAs for hepatitis C antibody detection and stability under different storage conditions

This study was undertaken to optimize and compare the efficiency of two commercial EIAs for anti‐HCV detection (HCV Ab Radim, Pomezzia, Italy and ETI‐AB‐HCVK‐4 DiaSorin, Vercelli, Italy), in dried blood spot (DBS) samples. The long‐term stability of anti‐HCV on DBS samples stored at three environmental conditions was also evaluated at: 2–8°C, 20–25°C, and −20°C. Paired DBS and serum samples were obtained from individuals with or without anti‐HCV. The type of elution buffer, sample and conjugate volume, sample incubation time and cut‐off values were evaluated. For both EIAs, a larger sample volume was used, and the cut‐off value determined by the manufacturer was employed for Radim EIA; however, ROC curve analysis was used for the DiaSorin EIA. The sensitivity and specificity of Radim EIA on DBS were 97.5% and 99.5%, respectively, and of DiaSorin EIA were 88.9% and 98.9%, respectively. Accurate results were obtained for a period of 117 days using DBS samples stored at all storage conditions, but storage at −20°C resulted in the lowest variation among the absorbance values. Both EIAs demonstrated the same limit of detection (until dilution of 1:104 with estimated viral load of 3.1 × 10−1 UI/ml), but the Radim EIA was associated with the best performance because a low coefficient of variation was observed in the repetition and reproducibility studies. In conclusion, commercial EIAs can be optimized for anti‐HCV detection in DBS samples that are extremely stable at different conditions for more than 100 days. J. Med. Virol. 84:1600–1607, 2012.